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1. 8 03 17R 25 8033622780120 EN doc 7 INTRODUCTION Mycobacteria are gram positive bacilli alchool acid resistant asporigenic strictly aerobics or microaerophilic motionless The Mycobacterium genre encompasses the Mvcobacterium tuberculosis complex and more than 80 species of non tubercular mvcobacteria atvpical which include pathogen species opportunistic and non pathogen species The most important species of Mvcobacterium genre is M tuberculosis the aetiological agent of tuberculosis one of the most important infectious diseases in the world which counts 8 millions of new cases and more than 2 millions of deaths everv vear Dve etal 1999 The most diffused and known form of tuberculosis is the pulmonarv disease even if almost each organ could be involved M tuberculosis is an intracellular pathogen that during the infection involves macrophages and monocvtes of peripheral blood macrophages of peripheral tissues promonocvtes and monocvtes of bone marrow In immunocompetent subjects the mycobacterium macrophage interaction leads to the activation of the same macrophage with a consequent bacterial Ivsis caused by processes of enzymatic digestion and oxidation To the contrary in immunocompromised subjects the inability to activate a bactericidal action is considered the main cause of Mycobacterium systemic diffusion As regards the pathogenicity mechanisms and the virulency factors the molecular data report the involve
2. absence of unspecific alignment and it has guaranteed the amplification of Mycobacterium tuberculosis Cross reactions with genomic DNA or other pathogenic microorganism nucleic acid have not been revealed 14 2 Diagnostic significance The increase of tuberculosis incidence and the development of tubercular strains antibiotic resistant request a fast and precise diagnostic method Generally for the diagnosis of tubercular disease also the isolation of only one bacillus results to be clinically significant 14 3 Diagnostic and analytic sensitivity Some bibliographical references have demonstrated that PCR is a very sensitive system able to find 10 tubercular bacillus in a population of 10 cells Its diagnostic sensitivity is about 84 pag 23 BANALITICA 03 17R 25 8033622780120 EN doc 15 BIBLIOGRAPHIC REFERENCES Dye C Scheele S Dolin P Pathania V Raviglione MC JAMA 282 677 86 1999 Wayne LG and Sramek HA Clin Microbiol Rev 5 1 25 1992 Horsburgh CR N Engl J Med 324 1332 1338 1991 Saiki RK Scharf S Faloona F Mullis KB Horn GT Erlich HA and Arnheim N Science 230 1350 1354 1985 Pai S Esen N Pan X Musser JM Arch Pathol Lab Med 121 859 864 1997 Riggio MP Gibson J Lennon A Wray D McDonald DG Gut 41 646 650 1997 Gulletta E Microbiol Med 10 2 41 44 1995 Niyaz A Mohanty AK Mukhopadhyay U Batish VK Grover S J Clin Microbiol 36 10 3094 3095 1998 Wang S X e Tay L Jour o
3. Repeat the DNA extraction from a smaller amount of clinical sample Use an adequate extraction system 3 Presence of aspecific products or extrabands after the visualization of the amplified products in agarose gel e The thermalcycler makes temperature changes too slowly Do a thermalcycler revision e The preparation of the amplification reaction has been executed in a long time at room temperature Accelerate the work time at room temperature Work on ice e The extracted has not been purified Use an extraction system which allows a good sample purification e The starting sample contained degraded DNA Repeat the extraction step using another clinical starting sample Be sure that the sample had been collected and stored in appropriate Way For any further problem contact AB ANALITICA technical support e mail laboratorio abanalitica it nautica pag 22 03 17R 25 8033622780120 EN doc 13 DEVICE LIMITS The kit can have reduced performances if the clinical sample is not suitable for this analysis using of alternative fixatives instead of neutral formalin buffer not correct sample storing the DNA is not amplifiable because of the presence of inhibitors of the amplification reaction or due to an inadequate extraction system The kit has not been stored at the suggested temperature 14 DEVICE PERFORMANCES 14 1 Specificity Primer sequence alignment in the most important databanks shows the
4. amplification are pre mixed and aliquoted in monodose test tubes 0 2 or 0 5 mL to which Taq polymerase and the extracted DNA will be added This premix format allows the reduction of the manipulation steps in pre amplification with considerable time saving for the operator the repeated freezing thawing of reagents that could alter the product performances is avoided and above all this form minimizes the risk of contamination so the risk to get false positive results Nevertheless it s always recommended to use all the proper amplification controls 10 COLLECTION MANIPULATION AND PRE TREATMENT OF SAMPLE The samples used for the determination of Mycobacterium tuberculosis infection by microbiological analysis and molecular biology are respiratory specimens saliva sputum expectorates bronchial secretions obtained by bronchoscopv bronchoalveolar washings and other kinds pleuric essudates pulmonarv Ivmphonodal and cutaneous and pleuric biopsies peripherial blood gastric aspirates that contain swallowed excretions liquor and bone marrow as well as cultures Riggio et al 1997 Gulletta 1995 pag 11 BANALITICA 03 17R 25 8033622780120 EN doc Generally given that tuberculosis is a disease with aerogenic infection the infection risk is correlated to the possibility of transmission of the micro organism present in air Some of the laboratory activities such as centrifugation stirring are more risky therefore
5. those operations must be performed with great caution It is very important to use vertical laminar flow cabinet during manipulation of Mycobacterium tuberculosis use disposable loops for collection of cultured strains and autoclave the waste materials 10 1 Respiratory specimens For safety during collection of the biological specimen it is a good rule to consider every clinical sample as potentially infectious and handle it as such Expectorate collection and bronchoscopy that could cause diffusion of infected mycobacteria in the environment should be done in suitable rooms and the staff should use an appropriate protection barrier The respiratory samples excreta could be fluidified by N acetil L cysteine 1 5 or dithiothreitol 0 1 the fluidifier should be added in equal volume with the excretum directly on the collection box and left at 37 C for 15 30 min according to the material consistency Generally respiratory samples are decontaminated by adding 4 NaOH solution then after centrifugation neutralized with HCI The obtained pellet could be resuspended in PBS The collection boxes should be sterile with screw cap and disposable To have a significant exam the sample should not be saliva but should be a sample from deep respiratorv tract Fresh or decontaminated samples should be stored at 2 8 C for short time not more than 48 hours or at 20 C for a longer period up to some months 10 2 Histological samp
6. use any part of the kit if over the expiration date e In case of any doubt about the storage conditions box integrity or method application contact AB ANALITICA technical support at laboratorio Dabanalitica it In the amplification of nucleic acids the investigator has to take the following special precautions e Use filter tips e Store the biological samples the extracted DNA positive control included in the kit and all the amplification products in different places from where amplification reagents are stored e Organise the space in different pre and post PCR units do not share nautica pag 4 03 17R 25 8033622780120 EN doc consumables pipets tips tubes etc between them e Change the gloves frequently e Wash the bench surfaces with 5 sodium hvpochloride e Thaw the PCR premixes at room temperature before use Add the Taq DNA polymerase and purified DNA very quickly at room temperature or in an ice bath 4 SAFETY RULES 4 1 General safety rules e Wear disposable gloves to handle the reagents and the clinical samples and wash the hands at the end of work e Do not pipet with mouth e Since no known diagnostic method can assure the absence of infective agents it is a good rule to consider every clinical sample as potentially infectious and handle it as such e All the devices that get directly in touch with clinical samples should be considered as contaminated and disposed as such In case of acc
7. 0 uL It is important to include in each experiment a negative control to monitor the contamination add distilled water to the mix instead of extracted DNA and a positive control any genomic DNA 11 2 2 M tuberculosis DNA amplification Add to each premixed test tube colourless tubes AB Taq 0 5 uL Extracted DNA 20 uL It is important to include in each experiment a negative control to monitor the contamination add distilled water to the mix instead of extracted DNA and 20 uL of positive control included in the kit pag 15 Danaumica 03 17R 25 8033622780120 EN doc Centrifuge shortly and put the B globin test tubes in the thermalcycler programmed as below 1 cycle 94 C 5 min 94 C 1 min 40 cycles 56 C 1 min 72 C 1 min Amplification fragments length of B Globin 268 bp Centrifugate shortly and put the M thermalcycler programmed as below tuberculosis test tubes in the 1 cvcle 94 C 5 min 94 C 2min 40 cycles 68 C 2 min 72 C 2 min Amplification fragments length of M tuberculosis 122 bp Banana pag 16 03 17R 25 8033622780120 EN doc 11 3 VISUALIZATION OF AMPLIFIED PRODUCTS 11 3 1 Agarose gel electrophoresis Preparation of a 3 agarose gel Weight 1 5 g of Agarose and pour it into 50 mL of 1X TAE Leave the solution on a magnetic stirring heater or in a microwave until the solution becomes clear Allow the gel to cool to nand warm a
8. ANALITICA ADVANCED BIOMEDICINE www abanalitica it USER MANUAL REF 03 17A Kit for the identification of DNA of Mycobacterium tuberculosis Ce 1 KIT CONTENT 3 2 STORAGE AND STABILITY OF REAGENTS 4 3 PRECAUTIONS FOR USE 4 4 SAFETY RULES 5 4 1 General safety rules 5 4 2 Safety rules about the kit 6 5 1 Reagents 7 5 2 Instruments 7 5 3 Materials 7 7 INTRODUCTION 9 8 TEST PRINCIPLE 10 10 COLLECTION MANIPULATION AND PRE TREATMENT OF SAMPLE 11 10 1 Respiratorv specimens 12 10 2 Histological samples 12 10 3 Blood 13 10 4 Liquor 14 11 AMPLIFICATION PROTOCOL 14 11 1 DNA EXTRACTION 14 11 2 DNA AMPLIFICATION 15 11 2 1 f Globin DNA amplification 15 11 22 M tuberculosis DNA amplification 15 11 3 VISUALIZATION OF AMPLIFIED PRODUCTS 17 11 3 1 Agarose gel electrophoresis 17 11 3 2 Sample loading 17 11 3 3 Interpretation of the results DNA amplification of R Globin and M tuberculosis 19 12 TROUBLESHOOTING 21 13 DEVICE LIMITS 23 pag 1 BANALITICA 03 17R 25 8033622780120 EN doc 14 DEVICE PERFORMANCES 23 14 1 Specificity 23 14 2 Diagnostic significance 23 14 3 Diagnostic and analytic sensitivity 23 15 BIBLIOGRAPHIC REFERENCES 24 INFORMATION FOR ORDERS 25 15 2 Related products 25 BANALITICA pag 2 03 17R 25 8033622780120 EN doc BOX P DESCRIPTION LABEL 8 test Monodose premix tube MB Colourl
9. M tuberculosis 3 MW DNA Marker aramos pag 20 03 17R 25 8033622780120 EN doc 12 TROUBLESHOOTING 1 Neither amplification products nor positive control DNA band e TAQ polymerase was not correctly added to the premix Use pipets and tips with suitable volumes pipet range 0 2 2 pL Check visually that TAQ polymerase diffuses in the premix this is easy because the enzyme is dissolved in glycerol that has a higher density alternatively check visually the drop of TAQ polymerase put on the tube wall then centrifuge briefly e The thermalcycler was not correctly programmed Check the conformity of the thermalcycler program and the temperature profile in the instruction manual then repeat the amplification with the correct program e The kit doesn t work properly Store the premix TAQ polymerase and positive control at 20 C Avoid repeated freezing thawing of the premix and the reagents 2 No amplification bands nor for 8 globin neither M tuberculosis in the tested sample but a good band for positive controls e Problems during the extraction step Be sure that the extraction kit is adequate and that you followed correctly all the instructions Consult the troubleshooting section of the extraction kit Repeat the DNA extraction starting from a new sample pag 21 BANALITICA 03 17R 25 8033622780120 EN doc e The amplification was inhibited Dilute the starting sample with distilled water and TE
10. NA molecules which correspond to the target sequence The sensitivity of this test makes it particularly suitable for the application in laboratory diagnostics Moreover the amplification reaction can be executed from a wide range of biological samples and since it allows to amplify very small DNA fragments the starting DNA can be also partially degraded Dinamica pag 10 03 17R 25 8033622780120 EN doc 9 PRODUCT DESCRIPTION The method of the MB kit consists of the amplification of a highly conserved and repeated region of the Mycobacterium tuberculosis genome the insertion sequence 18986 186110 This sequence is generally present in 1 to 20 copies for each bacteria The method also allows to evaluate the suitability of extracted DNA for the amplification by amplification of the B globin gene amplification control A negative result in B globin gene amplification indicates either the presence of inhibitors of the amplification reaction in the extracted DNA or that the DNA is highly degraded This method helps the operator to recognize possible false negative results The kit also provides with positive controls of amplification When the amplification of the positive control is successful it is guaranteed that the reaction is correct These controls are not dangerous for the operator because they are plasmid DNA containing only a part of Mycobacterium tuberculosis genome MB kit is in premix format all the reagents for the
11. ch could be the best material in case of extrapulmonary or scattered tuberculosis Sample collection should follow all the usual sterility precautions and should be transported in sterile boxes without transport medium Blood should be treated with EDTA Other anticoagulating agents as heparin are strong inhibitors of TAQ polymerase and so they could alter the efficiency of the amplification reaction Fresh blood can be stored at 2 8 C processed within 4 hours from the collection if DNA is not shortly extracted the sample must be frozen at 20 C A further step for lymphocytes isolation with a Ficoll Hypaque system or an erythrocyte lysis protocol is suggested pag 13 Danaumica 03 17R 25 8033622780120 EN doc 10 4 Liquor Also for this clinical specimen sample collection should follow all the usual sterility precautions and should be transported in sterile boxes with transport buffer 1X PBS addded In this case decontamination is not necessary Collect the biggest possible volume Fresh liquor can be processed immediately after the collection otherwise it can be stored at 2 8 C if processed within 4 hours from the collection Freezing the sample is not suggested 11 AMPLIFICATION PROTOCOL 11 1 DNA EXTRACTION For DNA extraction from respiratory specimens microbial culture in solid or liquid medium and histological samples AB ANALITICA suggests to use CLEAN MYC kit AB ANALITICA cod 04 40 with which t
12. ess T 25 50 8 Monodose premix tube BG Blue T 25 50 8 Taq DNA polvmerase AB TAQ termostable 5 UluL Red 1x37 5uL 1xX 75 ul 1 x 12 pl SMALL BAG Plasmidic DNA containing a part Positive control Blue 1 x 60 pL 1x 100 uL 1x20 pL of mycobacteria genome MB BOXF Electrophoresis Buffer for sample loading Blu 6X Blue 1x150uL 1x300uL 1x50uL Ethidium Bromide solution ma 2 5 mg mL oe Red 1x150uL 1x 250 pL 1x 100 uL S 36 3 45 DNA molecular weight marker MW Marker Yellow 1x150uL 1x 260 uL 1 x 50 uL BOX A Agarose for molecular biology AGAROSE 1x15g 1x27g 1x5g Electrophoresis Buffer TRIS Acetate EDTA pH 8 00 TAE 50X 1x50mL 1x100mL 1x20mL pag 3 ANALITICA 03 17R 25 8033622780120 EN doc ADVANCED DIOMEDICINE www abanalitica it 2 STORAGE AND STABILITY OF REAGENTS Each component of the kit should be stored according to the directions indicated on the label of the single boxes In particular Box P store at 20 C Small bag store at 20 C Box F store ata 2 8 C Box A store at 15 25 C When stored at the recommended temperature all test reagents are stable until their expiration date 3 PRECAUTIONS FOR USE e The kit should be handled by investigator qualified through education and training in molecular biology techniques applied to diagnostics e Before starting the kit procedure read carefully and completely the instruction manual e Keep the product out of heating sources e Do not
13. f Clin Microb 1932 1934 June 1999 Folgueira L Delgado R Palenque E Aguado J M Noriega A R Jour of Clin Microb 512 515 Mar 1996 Yam W C Yuen K Y Seto W H Clin Chem Lab Med 36 8 597 599 Aug 1998 Afghani B Stutman H R Biochem Mol Med 57 1 14 18 Feb 1996 15 1 Useful web sites http www medicalsystems it editor Caleidoscopio CalPDF 51 cal pdf nautica pag 24 03 17R 25 8033622780120 EN doc INFORMATION FOR ORDERS MB Kit for the identification of DNA of Mycobacterium tuberculosis cod 03 17A The kit contains the reagents for DNA amplification and visualization by agarose gel electrophoresis the internal control of sample amplificability and positive control Target region insertion sequence 156110 18986 Cod Product Pkg 03 17A 25 MB Mycobacterium tuberculosis 25 tests 03 17A 50 MB Mycobacterium tuberculosis 50 tests MB Kit for the identification of DNA of Mycobacterium tuberculosis cod 03 17R The kit contains the reagents for DNA amplification the internal control of sample amplificability and positive control Target region insertion sequence 156110 18986 Cod Product Pkg 03 17R 25 MB Mycobacterium tuberculosis 25 tests 03 17R 50 MB Mycobacterium tuberculosis 50 tests 15 2 Related products CLEAN MYC Kit for quick Mycobacteria DNA extraction and purification with a spin column method without use of phenol or chloroform from fresh frozen and formali
14. he kit has been standardized This method involves the use of Proteinase K and Lisozyme bacteriolityc enzymatic protein able to break the glicosidic B 1 4 bonds responsible for wall rigidity The two solutions allow the demolition of the wall surrounding the mycobacterium that makes difficult the DNA extraction Other DNA extraction methods can be used In particular some good results have been obtained with an extraction system which causes cellular lysis by repeated steps of freezing boiling During this steps it could be useful to add Tris EDTA and Chelex 100 to the sample these solutions stabilize the DNA during the boiling step keeping the ionic force of the sample In literature it is described the combination of a first step of freezing boiling with bacteria lysis by Lisozyme and Proteinase K digestion Niyaz et al 1998 In any case it is necessary to remember that the volumes of the extract indicated in paragraph 11 2 1 and 11 3 1 for B globin and Mycobacterium tuberculosis amplification 20 uL are referred to extracts obtained with AB ANALTICA methods Using an alternative extraction method if the volume of the DNA solution to amplify is less than 20 uL it will be necessary to adjust the volume of the amplification mix by adding water nautica pag 14 03 17R 25 8033622780120 EN doc 11 2 DNA AMPLIFICATION 11 2 1 B Globin DNA amplification Add to each premixed test tube blue tubes AB Taq 0 2 uL Extracted DNA 2
15. idental spilling of the samples clean up with 10 Sodium Hypochloride The materials used to clean up should be disposed in special containers for contaminated products e Clinical samples materials and contaminated products should be disposed after decontamination by immersion in a solution of 5 Sodium Hypochloride 1 volume of 5 Sodium Hvpochloride solution every 10 volumes of contaminated fluid for 30 minutes OR autoclaving at 121 C at least for 2 hours NOTE do not autoclave solutions containing Sodium Hypochloride pag 5 BANALITICA 03 17R 25 8033622780120 EN doc 4 2 Safety rules about the kit The risks for the use of this kit are related to the single components Dangerous components 3 8 diamino 1 ethyl 6 phenylphenantridiumbromide Ethidium Bromide lt 2 Description of risk T Toxic 8 RISK SENTENCES AND S SENTENCES ETHIDIUM BROMIDE R23and R68 Toxic for inhalation Risk of irreversible effects S 36 37 45 Wear laboratory coat and disposable gloves In case of accident or discomfort seek for medical assistance and show the container of label R and S sentences refer to the concentrated product as provided in the kit In particular for Ethidium Bromide until the dilution in the agarose gel In manipulating concentrated Ethidium Bromide use a chemical dispensing fume cabinet Always wear disposable gloves and laboratory coat in manipulating the diluted Ethidium solution as well The prod
16. les Histological samples are pulmonary skin lymphonodal and pleural biopsies fresh frozen formalin fixed and paraffin embedded Perform the bioptical sample collection as routine Fresh biopsies can be treated within few minutes from sampling or quickly frozen with liquid nitrogen and successively stored at 80 C until mechanic disgregation by using a sterile cutter followed by enzymatic digestion nautica pag 12 03 17R 25 8033622780120 EN doc In case that the biopsy is fixed and paraffine embedded it is suggested the use of formalin buffered at pH 7 with sodium and potassium salts at 10 as Lilie formula Tissue fixation with not buffered formalin in Bouin Holland or other acidic fixatives osmic acid for istance are not suitable for subsequent DNA extraction because that substances produce cross links in the tissue making it not digestible In case of fresh or frozen histological samples up to about 50 mg it is suggested to proceed immediately with the mechanic disgregation of tissue by using a sterile cutter Do this operation on a glass slide adding an aliquote of 1X PBS buffer Transfer the minced tissue in a tube by a Pasteur pipette and proceed with sample digestion If the histological sample is formaline fixed and paraffine embedded proceed with paraffin removal and then with the enzymatic digestion 10 3 Blood The Mycobacterium tuberculosis searching could be done starting from peripheral blood whi
17. ment of the elements that characterize the structure of the bacterial wall of the micro organism lipoarabinomannan sulfatide and proteins in particular for the activation of cell mediated immunitary response The sulfatide seems to be the first responsible for macrophage activation and for the increased secretion of IL 18 and TNF a favouring the formation of granulomas that slowly develop giving a very extended tissutal destruction The observed hystopathological lesions could be exudative lesions characterized by acute inflammation exudate formation and accumulation of PMN leucocytes around the bacilli and productive lesions granulomatous characterized by concentric distribution of macrophages epithelioid cells to form tubercles and giant cells Even though the initial diagnosis of Mycobacteria infection is often based on clinical data the definitive diagnosis requires isolation and identification of the micro organism in a laboratory The classic laboratory procedures for clinical samples analysis involve the decontamination and digestion of the specimen the direct microscopic detection of the presence of AFB acid fast bacilli after staining of the slides prepared with infectious materials with Ziehl Neelsen the staining with rhodamine auramine fluorescent mix the isolation of the micro organism in pag 9 BANALITICA 03 17R 25 8033622780120 EN doc culture and test of drug resistance Due to the reduced growing speed
18. n fixed and paraffin embedded histological specimens respiratory specimens and microbial cellular cultures in solid or liquid soil Cod Product Pkg 05 40 50 CLEAN MYC 50 tests 05 40 100 CLEAN MYC 100 tests pag 25 BANALITICA 03 17R 25 8033622780120 EN doc ANALITICA AB ANALITICA srl Via Svizzera 16 35127 PADOVA ITALY S Tel 39 049 761698 Fax 39 049 8709510 e mail info abanalitica it
19. nd then add 10 uL of Ethidium Bromide solution NOTICE Ethidium Bromide is a strong mutagenic agent always wear gloves and preferably work under a chemical safety cabinet during the handling of this reagent or gels containing it Place the gel into the appropriate gel casting tray with the comb placed in and allow the gel to cool at room temperature or in a fridge until the gel becomes solid When the gel is solidified remove carefully the comb pay attention not to damage the gel wells transfer the tray into the electrophoresis chamber and pour the appropriate amount of TAE buffer so that it covers completely the gel about 1 2 mm over the gel surface 11 3 2 Sample loading Mix into a tube or directly on a parafilm layer 2 uL of 6X Blue 10 uL of PCR product or DNA molecular weight marker MW Marker Load the mixture on the gel wells switch on the power supplv and set the voltage between 80 100 V Run the gel for about 30 45 min then place the gel on an UV transilluminator and analvze the results bv comparing the size of the amplification products with the reference Molecular Weight Marker DNA Molecular Weight Marker Marker MW 501 489 404 353 242 190 147 110 89 67 34 26 bp NOTE In a 3 agarose gel the 501 489 bp bands are usually not clearly pag 17 BANALITICA 03 17R 25 8033622780120 EN doc resolved and appear as an unique band the 26 and 34 bp bands are sometimes too small to be visible in a 3 agar
20. of Mycobacteria their isolation requires some weeks at least 3 9 weeks with optimum temperature at 37 C In the last ten years different molecular methods for direct identification of Mycobacteria have been developed The identification of Mycobacterium tuberculosis with classical methods based on the phenotype on the biochemical characteristics and on the use of chromatographic techniques requires long execution time not suitable for routine screening PCR based methods allow to get the same information with less time and costs Stauffer et al 1998 Glennon et al 1994 8 TEST PRINCIPLE PCR method Polymerase Chain Reaction has been the first method of DNA amplification described in literature Saiki RK et al 1985 It can be defined as an in vitro amplification reaction of a specific part of DNA target sequence by a thermostable DNA polymerase Three nucleic acid segments are involved in the reaction double stranded DNA template to be amplified target DNA and two single stranded oligonucleotides primers that are designed in order to anneal specifically to the template DNA The DNA polymerase begins the synthesis process at the region marked by the primers and synthesizes new double stranded DNA molecules identical to the original double stranded target DNA region by facilitating the binding and joining of the complementary nucleotides that are free in solution dNTPs After several cycles one can get millions of D
21. ose gel because of their low molecular weight NOTICE UV rays are dangerous for skin and above all eyes always wear gloves and safety glass or make use of the protection screen of the UV transilluminator nautica pag 18 03 17R 25 8033622780120 EN doc 11 3 3 Interpretation of the results DNA amplification of B Globin and M tuberculosis The included controls should show the following results CONTROL RESULT INTERPRETATION Positive Control present The PCR amplification is correct Negative Control absent Absence of contaminations Then the interpretation of the bands on agarose gel follows the table below BAND RESULT INTERPRETATION Sample not suitable for B Globin band absent amplification repeat the DNA extraction B Globin band present Amplificable sample M tuberculosis band absent M tuberculosis negative B Globin band present Amplificable sample M tuberculosis band present M tuberculosis positive It is possible that positive samples show aspecific bands this could depend on the type of starting sample for example paraffin embedded material but it will not interfere with the analysis For any further information contact AB ANALITICA technical support e mail laboratorio abanalitica it 03 17R 25 8033622780120 EN doc pag 19 BanaLimon 122 Fig 1 3 agarose gel electrophoresis 1 Positive sample for M tuberculosis 2 Positive sample for
22. uct can not be disposed with the common waste It must not reach the drainer system For the disposal follow the local law In case of accidental spilling of Ethidium Bromide clean with Sodium hypochloride and water Safety data sheet MSDS of Ethidium Bromide is available upon request Dinamica pag 6 03 17R 25 8033622780120 EN doc 5 MATERIALS REQUIRED BUT NOT PROVIDED 5 1 Reagents Sterile DNase and RNase free water Distilled water 5 2 Instruments Laminar flow cabinet use is recommended while adding TAQ polymerase to the amplification premix to avoid contamination it would be recommended to use another laminar flow cabinet to add the extracted DNA Micropipettes range 0 2 2 uL 0 5 10 pL 2 20 uL Thermalcycler Microcentrifuge max 12 000 14 000 rpm Balance Magnetic heating stirrer or microwave Chemical cabinet use is recommended in handling Ethidium Bromide Horizontal electrophoresis chamber for agarose minigel Power supply 50 150 V UV Transilluminator Photo camera or image analyzer 5 3 Materials Disposable gloves Disposable sterile filter tips range 0 2 2 uL 0 5 10 uL 2 20 uL Graduate cilinders 1 L for of TAE dilution Pyrex bottle or Becker for agarose gel preparation Parafilm pag 7 BANALITICA 03 17R 25 8033622780120 EN doc 6 PREPARATION OF REAGENTS Preparation of 1 Lof 1X TAE buffer Mix 20 mL of 50X TAE with 980 mL of distilled water nautica pag

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